The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function

Abstract

The von Willebrand factor (VWF) A2 crystal structure has revealed the presence of a rare vicinal disulfide bond between C1669 and C1670, predicted to influence domain unfolding required for proteolysis by ADAMTS13. We prepared VWF A2 domain fragments with (A2-VicCC, resi- Introductionvon Willebrand factor (VWF) forms disulfide-linked multimers, the largest of which are most potent in binding collagen and the platelet receptor glycoprotein Ib␣. [1][2][3][4] Mechanical shear forces in the bloodstream induce conformational changes in VWF 5 and modulate the exposure of both platelet and ADAMTS13-binding sites. VWF multimer size and function are regulated through proteolysis by ADAMTS13. 6,7 Unfolding of the central VWF A2 domain is required to expose the Y1605-M1606 cleavage site, which is normally buried within the central ␤-sheet of the domain. 8 The VWF A2 domain is highly homologous to the VWF A1 and A3 domains (supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) but lacks an intradomain disulfide bond that connects the ␤1-sheet with the ␣6-helix. However, the recent VWF A2 domain crystal structure revealed a disulfide bond between adjacent C1669 and C1670 at the C-terminus of the ␣6-helix. 8 Such a vicinal disulfide bond is rare, as an 8-membered ring is formed that bends the protein backbone in an unusual constrained conformation. 9 In the crystal structure, the disulfide bond directly interacts to the hydrophobic core of the domain, suggesting that it stabilizes the domain conformation. 8 We therefore examined the influence of this unusual vicinal disulfide bond on VWF A2 domain function. Methods Recombinant proteinsVWF A2-⌬CC (amino acids 1473-1668), A2-VicCC (1473A2-VicCC ( -1670, and both N1493C/C1669G (A2-CC1) and N1493C/C1670G (A2-CC2) mutants (supplemental Figure 1B) were cloned in the pCEP4 vector (Invitrogen) containing a C-terminal myc/His tag. A2 domain fragments were expressed in HEK293EBNA cells, purified by Ni 2ϩ chromatography, and quantified by the BCA protein assay kit (Thermo Scientific).Mutations N1493C/C1669G (VWF-CC1), N1493C/C1670G (VWF-CC2), and C1669G/C1670G (VWF-VicGG) were introduced into pcDNA3.1-VWF 10 and full-length VWF variants expressed in HEK293EBNA cells. Conditioned media were dialyzed and VWF concentrations determined by enzyme-linked immunosorbent assay. 10 AD-AMTS13 was expressed and purified as previously described. 11 Maleimide-PEG 2 -biotin labeling of free thiolsFree thiols were detected both before and after reduction of VWF A2 variants by labeling with maleimide-PEG 2 -biotin (Thermo Scientific). Labeled thiols were detected by Western blotting using streptavidin-peroxidase. Reduction and carboxymethylation of VWF A2 variantsTo permanently disrupt disulfide bonds, VWF A2 variants were reduced with dithiothreitol (DTT) and carboxymethylated with iodoacetic acid before use in the proteolysis and binding assays. Proteolysis by ADAMTS13Proteolysis of VWF A2 variants was performed as previously de...

Mechanisms by which von Willebrand Disease Mutations Destabilize the A2 Domain*

“…In this mutation, the long, unbranched, hydrophobic Met-1528 side chain is replaced the smaller, ␤-branched, hydrophobic Val side chain. Met-1528 at the end of the ␣1-helix is buried by the critical vicinal disulfide bond between Cys residues 1669 and 1670 (21) and by Leu-1666. These three residues are at the end of the ␣6-helix and critically stabilize the C-terminal end of VWF A2 against applied elongational force.…”

Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13)

“…A critical feature of VWF unfolding has been shown to be the unfolding of the A2 domain itself. This domain is stabilized by a hydrophobic plug comprised of vicinal Cys residues located at the C terminus of the domain, which is inserted in the center of the domain near to the scissile bond (3,20). This hydrophobic plug must first be removed for the A2 domain.…”

N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance

“…This cysteine clamp mutation has previously been described to prevent A2 domain unfolding and render VWF insensitive to ADAMTS13 cleavage. 45,46 In vitro modification of VWF glycan structures…”